1. Field of the Invention
The present invention generally pertains to chemical or elemental markers or tracers that, when combined with other chemical admixtures or compounds, or when inserted into or upon objects or devices, thereafter serve to permanently identify such admixtures, compounds, objects or devices, including after such change(s) and gross change(s) to the compounds, objects or devices in form and/or in composition as may be occasioned by lapse of. time, dissipation, wear, deterioration, oxidation, or explosion.
The present invention particularly concerns (i) elemental or chemical markers that are indefinitely long lasting, and detectable at the level of a single atom or molecule over a fast range of densities; (ii) the use of neutron activation in the detection of elemental and chemical markers, and the elemental or chemical markers so detectable; and (iii) the packaging of, and/or carriers for, neutron-activation-detectable elemental and/or chemical markers, including the packaging of elemental and/or chemical markers in carrier microspheres, including microspheres as are used in biological experimentation including, inter alia, in blood flow analysis.
2. Description of the Prior Art
2.1 Neutron Activation Analysis
The present invention will be seen to employ neutron activation analysis.
Individual stable elements (e.g., gold) are known to have isotopes that are strongly detectable by neutron activation analysis. The known abundance of the "marker" elements in certain substances has permitted these elements to serve as markers for the substances. See Kennelly, J. J., Apps, M. J., Turner, B. V. and Aherne, F. X. 1980; Dysprosium, cerium and chromium marker determination by instrumental neutron activation analysis. Can. J. Anim. Sci. 60:749-761. See also Nishiguchi Y., Sutoh M., Nishida T., Satoh H., Miyamoto S.; Neutron activation analysis of Lanthanides (La, Sm and Yb) as a particle marker, and estimation of passage rates. Anim. Sci. Technol. (Jpn.), 67: 787-793. 1996.
Neutron activation analysis can provide the life science community with capabilities not readily available with other assay technologies. Neutron activation is well known for both (i) its excellent sensitivity and (ii) its specificity for the simultaneous measurement of multiple elemental trace elements. This specificity offers the potential of being able to measure multiple isotopic tracers per assay.
Unlike light, neutrons can penetrate solid tissue and opaque-liquid samples, thereby providing an assay that is completely self-contained with but minimal sample preparation.
Unlike other element detection methods, such as atomic absorption spectrophotometry, neutron activation is not chemically or physically destructive. Therefore, samples can be archived, re-assayed, and/or undergo additional chemical analysis following neutron activation.
Because only the samples of interest are neutron activated, assay by neutron activation analysis can significantly reduce-occupational exposure to radiation and eliminate the low-level radioactive waste generated. For example, the contamination of gloves and protective clothing, glass and plastic laboratory supplies, and waste products from research animal housing and carcasses attendant upon the use of radioactive tracers are completely avoided. Stable isotopes do not undergo further radioactive decay or cause radiokinistics and, unlike some colorometric probes, they do not suffer any loss of activity (i.e., loss of fluorescence) over time. Therefore, stable isotope labeled products will have an indefinitely long shelf life: significantly longer than competing labeling methods.
The major disadvantage to assay by neutron activation technology is the required access to a neutron source. If the neutron radiation source is to be strong (i.e., with a high flux) so that it will excite a significant proportion of the target tracers--preferably stable isotopes--of the sample--which sample may be sparse--to an excited, radioactive, energy level in a reasonable time, providing thereby a reasonable population of radionuclides the decay of which may likewise be detected during a reasonable time, then the source of neutron flux must most commonly be energetic, as is typically derived from a research reactor. Suitable research reactors, and reactor time, are not scarce in the United States circa 1999. However, the reactors are located at particular sites not normally coincident with sites at which investigations in the life sciences are conducted. Therefore, samples for assay by neutron activation analysis must normally be sent to a reactor, irradiated with neutron flux, and analyzed with results being reported to the sender. Furthermore, the samples, if not permanently archived at or near the site of the reactor, may be returned to the sender only when radiation has sufficiently abated.
Accordingly, and despite the many advantages of neutron activation, this general analytical tool has not (as of 1999) reached its full potential within the life science community due to some combination of (i) a lack of user awareness of the technique, (ii) a lack or perceived lack of access to reactors, and/or, importantly to the present invention, (iii) a lack of commercially available stable-labeled research products specifically designed for neutron activation technology. These research products would desirably be targeted on intended biological research applications, and be of a form familiar to biological researchers.
Most recently, BioPhysics Assay Laboratory, Inc., 280 Wellesley Avenue, Wellesley Hills, Mass. 02481 (Phone/Fax: (781) 239-0501) ["BioPAL"] has been formed to (i) develop, manufacture and market a new generation of high-precision stable-labeled research products, and to (ii) provide a state-of-the-art assay service that can meet commercial demand. The present invention will be seen to concern these stable-labeled research products, developed jointly with Triton Technology, Inc., of San Diego, Calif.
In passing, it should be noted that neutron activation analysis also has an extensive history in the detection of explosives--which is a different thing than the detection of stable isotopes, used as makers, that may be placed into, inter alia, explosives as will be taught by the present invention. In other words, certain chemicals present in certain explosives can be directly detected by neutron activation analysis. As a leading book on this topic, see Explosive detection using fast neutron activation analysis by Terry E. Carrell, published by North American Rockwell, Los Angeles, Calif.
When the present invention is later understood to be stable-isotope labeled microspheres usable as markers in diverse circumstances, it will be useful to consider the possible use of these markers in labeling explosives. Stable-isotope labeled microspheres serving as identifying markers cannot be assured to be emplaced in explosives save those legitimately produced, and then only under mandate of law or regulation.
However, in accordance with the general principles of the present invention, later explained, to the effect that the carriage, and the chemistry, of the marker stable isotope is divorced from the chemistry of the exterior surface of a microsphere which serves to mechanically retain the marker stable isotope, it can be anticipated, in advance, that ubiquitous, tailored, stable-isotope labeled microspheres in accordance with the present invention will be very useful for permanently marking an immense number of different admixtures, compounds, objects or devices, including, inter alia, explosives. Properly tailored to a target, including an explosive, about the only way of expunging microspheres and any elemental markers that they contain from a compound such as an explosive is by gross molecular dissociation such as is characteristic of, inter alia, explosion. Of course, to remove the microspheres, and markers, requires destruction of the compound. Moreover, such explosion and attendant molecular dissociation does not truly get rid of the elemental markers, which remain (and will remain, short of atomic transmutation) as residue.
It will next immediately be discussed that microspheres may be both (i) mechanically and/or (ii) chemically, tailored, or "targeted", to their intended environment of use. Most of this discussion will involve prior art biological uses of microspheres. Stable-isotope labeled microspheres will later be seen to be fully as susceptible of being "targeted" upon an intended environment of use as were previous microspheres. Moreover, and recalling the requirements of labeling explosives, it should be appreciated in considering the diverse labeling and marking requirements of the prior art (both biological and non-biological), that the "targeting" of stable-isotope labeled microspheres in accordance with the present invention on an intended environment of use will prove to be selectively either broad or narrow, and semi-permanent or permanent, all in accordance with (i) application requirements and (ii) the principles of chemistry.
2.2 Types of Microspheres, Circa 1999
The mechanism of the present invention for delivering stable elements that are suitable to form isotopes detectable by neutron activation analysis will be seem to be: microspheres.
"Microspheres" is the generic term applied to certain minute, typically homogeneous and uniformly-size-graded, particles, beads or whatever existing in over 2000 types. The microspheres are commonly made of latex rubber, polystyrene (PS) plastic, or other polymers, copolymers, terpolymers, and silica. They come in a variety of densities from 0.9-2.3 g/ml. They come in a sizes ranging from nanometers to millimeters.
Microspheres are both (i) mechanically and (ii) chemically versatile; properties of which good utility will later be seen to be made in the present invention.
A broad range of sizes and types or microspheres--also known as uniform latex particles--are available from commercial sources. For example, diverse microspheres are available from Bangs Laboratories, Inc., 979 Keystone Way, Carmel, Ind. 46032. Microspheres are available in diameters from .about.0.020 .mu.m (20 nm) to 1000 .mu.m (1 mm). Size uniformity is excellent: c.v.'s are typically &lt;3%, and are often .about.1%.
Colored and fluorescently-colored microspheres are available from said Bangs Laboratories, Inc., from Triton Technologies, Inc., and from Molecular Probes, Inc. in a spectrum of colors. More than 300 different dyed microspheres are available in colors from red to beyond violet, as well as black, white, and gray. Bright primary colors are available for easy color identification and mixing of colors; others are very dark, like ink, for good contrast.
Microspheres may be obtained that are dyed with fluorescent dyes. More than 30 different-sized microspheres colored with &gt;10 different absorbance and fluorescent dyes exhibiting various excitation and emission wavelengths are commercially available.
Microsphere surface chemistries range from hydrophobic (plain polystyrene) to very hydrophilic surfaces imparted by a wide variety of functional surface groups: 1) aldehyde --CHO; 2) aliphatic amnine --CH2--NH2; 3) amide --CONH2, 4) aromatic amine-NH2; 5) carboxylic acid --COOH (3 different types); 6) chloromethyl --CH2--Cl; 7) epoxy; 8) hydrazide --CONH--NH2; 9) hydroxyl --OH; 10) sulfate --SO4; and 11) sulfonate --SO3.
The original recipe for various types of .about.1 .mu.m (100 nm) diameter COOH-- or --NH2 modified microspheres contained 12, 20, 40, or 60% magnetite. Truly superparamagnetic, these microspheres respond to a magnet but display no residual magnetism. These microspheres can be used for direct adsorption. of proteins, or surface groups can be used for covalent coupling of ligands (proteins, DNA, etc.)
The narrow size distribution type were designed for better performance in cell depletion applications. Encapsulated microspheres have a magnetite-rich core and a polystyrene shell, with COOH and NH2 surface groups. These microspheres make better solid supports for applications using enzymes because there is no iron on the surface.
As an example of microspheres with attachments, the Pro active Streptavidin coated superparamagnetic beads of Molecular Probes, Inc., serve as a generic magr.et.ically responsive solid phase to which a variety of biotinylated items can be attached. The Pro active Protein A coated magnetic microspheres provide an IgG-binding affinity support that is extremely easy to manipulate.
For example, Pro active GAM magnetic microspheres having goat anti-mouse Fc-specific IgG serve to bind mouse IgG's and orient the mouse IgG's correctly for high activity with less- primary Ab.
Streptavidin, protein A, and GAM coated non-magnetic, polymeric microspheres are also available in several sizes. Some of the more common applications of protein-coated microspheres include: (i) affinity chromatography, (ii) multi-purpose solid phase for immunoassays, (iii) nucleic acid hybridization, (iv) immunoselective cell separation, and (v) DNA sequencing.
For purposes of the present invention, it should only be understood that microspheres can both (i) mechanically carry diverse elements within their matrix--for example, ferromagnetic iron--and can (ii) chemically affix diverse chemical compounds, including those of biological interest. Both the (i) mechanical and (ii) chemical combinations can be relatively permanent, essentially demanding a destruction of the microsphere (which may not be easy) in order to sever the association.
2.3 Uses Of Microspheres, Circa 1999
2.3.1 Blood Flow Analysis
Microspheres of both the radioactive and the non-radioactive, absorbance-dye and fluorescent-dye labeled, types are regularly used in the measurement and analysis of in vivo blood flow or, more particularly, regional myocardial blood flow (RMBF). Such microspheres are also used in measurement of the flow of gas in the lungs, or the movement of materials through the gut, or like fluid movement processes occurring within the higher animals.
As regards the use of radioactive-labeled microspheres, their presence is detected with detectors of gamma radiation emitted during decay events.
As regards the use of non-radioactive absorbance-dyed or fluorescently-dyed microspheres, the dyes used to mark the microspheres may be, in different variants, either elutable to non-elutable, with the dye absorbance or fluorescence measured in various ways, including in bulk by the use of automated or semi-automated absorbance or fluorescence detector equipments.
2.3.2 Latex Agglutination Tests and Particle Immunoassays
Many microspheres are used in various medical diagnostic applications. Proteins will adsorb readily onto polystyrene (PS) microspheres or they may be covalently coupled to. carboxylic acid or other surface functional groups. Microspheres so coated with antibodies can be agglutinated (agglomerated) by the appropriate antigens.
2.3.3 Sandwich Assays and Tests (Particle Capture ELISA's)
Antibody-coated microspheres form the basis for particle capture ELISA tests and related assays (i.e., those that form a blue dot). Antigen links the sandwich of (1) primary antibody-coated particle and (2) enzyme-labeled secondary antibody. Microspheres permit easy preparation of reagents (antibody coating of microspheres done in bulk, not on a membrane) and precise placement of Ab-coated microsphere spots on top of the filters.
2.3.4 Dyed Particle Sandwich or Chromatographic "StripTests"
Darkly dyed microspheres can eliminate the need for enzymes (and their attendant stability problems) in sandwich assays. Tests use dyed microspheres attached to one of the two sandwich antibodies. Small, antibody-coated microspheres move easily through the membrane in chromatographic-like assays; deeply dyed (some as dark as ink!), they bring enough color to the sandwich to completely preclude the use of enzymes. A wide variety of colors are available, including bright fluorescents.
2.3.5 NIST-traceable Standards
Calibrated uniform diameter microspheres for use as standards and controls for particle or cell counting and measuring instruments are available singly or in sets.
2.3.6 LDV/PIV Seeds
Microspheres have been used as seed in fluid flow streams for laser Doppler and particle image (and other) velocimetry measurements. In these applications they maybe dispersed in gas streams, wind tunnels, wave tanks, or ship tow-tanks. Silica microspheres permit use in combustion studies and other high temperature applications, too.
2.3.7 Other Applications
Other applications for nicrospheres include 1) blood cell simulation; 2) cell separation; 3) phagocytosis studies; 4) chemiluminescent assays; 5) column packing (non-porous); 6) density calibrators; 7) DNA probes/ PCR; 8) instrument standards; 9) fluidized beds; 10) magnetic resonance imaging; 11) model studies; 12) solid phase DNA sequencing; 13) spacers for flat panel displays; and 14) void sources for ceramics.
Applications for dyed microspheres include 1) stains; 2) adjuvants; 3) contrast agents; 4) cell tags (rosettes); 5) gel permeation markers; 6) flow markers for liquids; and 7) confocal microscopy standards.
2.4 Methods of Using and Measuring Microspheres, Circa 1999
General background to the present invention as regards the use of microspheres in biological measurements may be found in U.S. Pat. No. 5,230,343 for COLORED MICROSPHERES FOR MEASURING AND TRACING FLUID MIXING AND FLOW, PARTICULARLY BLOOD FLOW TO TISSUE to inventors Gerd Heusch, Michael P. Guberek, and W. Scott Kemper; in U.S. Pat. No. 5,253,649 for a PROCESS FOR THE MEASUREMENT OF BLOOD CIRCULATION BY MEANS OF NON-RADIOACTIVE MICROSPHERES to inventors Gerd Heusch, Rainer Gross, Wolfgang Paffhausen and Andreas Schade; and in German patent application Serial No. P 40 19 025.0 filed in Germany on Jun. 14, 1990 which is the priority application to both these patents. All these related patents are assigned to Triton Technology, Inc., of San Diego, Calif., a corporation of the State of California. The contents of the related patents are incorporated herein by reference.
2.4.1 Use of Microspheres in Fluid Flow Analysis
Traditionally, most measurements of fluid mixing and fluid(s) flow(s) are direct. One or more fluid flows may simply be measured while such flows are occurring. Alternatively, any mixture that results from the flows of two or more fluids may be analyzed as to its constituent components in order to quantitatively determine the fluid flows that have transpired.
However, direct measurement of fluid(s) flow(s), such as within the blood stream of a living animal, is often impossible. Moreover, direct quantitative analysis of the constituent components of complex, or extensive, mixtures of fluids is often prohibitively difficult or expensive. The expense is magnified if many samples must be taken, and analyzed, over time.
Accordingly, modeling or simulation is sometimes used in order to trace the flow, and mixing, of one or more fluids. For example, a dye may be put in ground water and its dispersion may subsequently be observed. From the observed dispersion of the dye a similar dispersion of pollutants, or other less readily detectable fluids, may be imputed.
Another, relatively sophisticated, form of fluid flow and fluid mixing analysis is indirect. A physical marker is put into, or a chemical marker is bonded to, an actual fluid, or a fluid component, for which flow and/or mixing is desired to be assessed. The fluid serves as a carrier. When the distribution of the marker is analyzed then the corresponding distribution of the carrier fluid is imputed.
The highest, and most exacting, expression of this indirect method is in medicine, and particularly in blood flow analysis. The blood, and the organs and tissues receiving blood, within a living animal present a system that is very complex in its fluid flow patterns and dynamics, and that is difficult of direct access and measurement. Accordingly, microscopic markers are placed by catheter into the left atrium of the animal's heart, entering into the animal's blood thoroughly mixed where they are subsequently distributed to the animal's tissues in proportion to the blood flowing to the tissues.
The microscopic markers are commonly microspheres sized (typically less than 30 .mu.m and more than 7 .mu.m and more typically 15 .mu.m) so that they are trapped by, and permanently lodge within, the smallest capillaries of the animal's tissues. In proportion to the flow of blood, the particles are sized so that they are trapped in the capillaries on their first pass through the circulation system of the animal. The tissues are subsequently harvested, and the prevalence--i.e., the numbers--of the markers have previously been analyzed, producing thereby an indirect indication of the blood flow to the tissue.
Previous systems developed for medical blood flow analysis--discussed in greater detail hereafter--have proven to be both complex and expensive. Because of their cost and complexity, such systems have not been found suitable for use in routine industrial or environmental fluid flow and mixing measurement problems.
However, it should be recognized that the flow of blood, or blood components, within the arteries and veins of a living animal is only different in complexity, and not in the essential nature of fluid flow dynamics, from the flows of fluids occurring within factories, ecosystems, and the like. Accordingly, if a reliable, effective, inexpensive, and automated (or semi-automated) indirect fluid flow measurement system suitable for use on the difficult problem of blood flow analysis could be developed, then such a system might well have general applicability to the tracing and measurement of fluid flows, and the mixing of fluids, in many other diverse applications.
2.4.2 The Earliest Measurements of Blood Flow with Radioactive Microspheres
The reasons for the measurement of blood flow are set forth in U.S. Pat. No. 4,616,658 to Shell, et. al., for NON-RADIOACTIVELY LABELED MICROSPHERES AND USE OF SAME TO MEASURE BLOOD FLOW. Shell and his co-inventor See teach a safe and inexpensive method of measuring blood flow in experimental animals using non-radioactively labeled microspheres is provided. The microspheres may be comprised of a variety of materials, including latex and agarose, and may be labeled with colored dyes or by linkage to enzymes, plant enzymes being preferred because they do not occur naturally in an animal's system. After injection and circulation of the microspheres throughout the animal's system, blood flow to particular tissue may be. measured by counting the number of microspheres in the tissue sample, the initial number of microspheres in the animal's blood stream having been measured shortly after injection. In the case of microspheres labeled with colored dyes, the spheres may be counted in tissue either after separation from the tissue or while still trapped in the tissue's capillaries. Techniques for separating the microspheres from blood and tissue are also provided.
The measurement of blood flow in experimental animals is often necessary in the fields of pharmacology, physiology, therapeutics and diagnostics. For example, toxicology studies require blood flow measurement to determine the toxicity of various suspected toxic agents. Further, many diagnostic and therapeutic advances have some impact on the flow of blood. It is therefore desirable to take blood flow measurements.
Blood flow measurements can be performed in many anatomical areas, including the brain, heart, lung, gut, kidney, reproductive organs, skin and muscle. One sensitive and specific previous technique involves the use of radioactively labeled microspheres. In one variant of the technique plastic or polystyrene microspheres are marked with a radioactive label and injected into the left atrium of the heart of an experimental animal. They are injected into the left atrium in order to achieve homogeneous mixing of the spheres in the blood. The prevalence of the radioactively-labeled microspheres in the blood is assessed by withdrawal of a blood sample from the aorta downstream from the heart, during the complete course of the atrial injection. This "reference withdrawal" sample is used to determine the "radioactivity per volume flow rate" of the blood coming from the heart. The mixed microspheres disperse in proportion to blood flow and lodge in the micro-capillaries within the tissues of the animal. The animal is later sacrificed and the organ(s) of interest is (are) harvested. Blood flow to a particular organ is determined by measuring the level of radioactivity in the organ sanple, which radioactivity is a function of the number of microspheres trapped in each portion of the organ. This radioactivity level is divided by the reference withdrawal value in order to determine absolute blood flow.
Notably, the radioactive strength, or intensity, of the injected microspheres is not required to be exactly known. Ultimately only ratios between the (i) density of injected microspheres, and (ii) the density of microspheres recovered from each tissue, will prove relevant. To start, blood is withdrawn at a predetermined rate from a site downstream from the point of injection for a longer time than it takes for all the injected microspheres to pass this point. Only a small fraction of the flowing blood, and a commensurately small fraction of the microspheres contained within the blood, are extracted. However, the density of the microspheres within the blood is directly determinable in terms of units radioactivity (i.e., radioactive intensity) per unit measure of blood flow rate (volume per unit time). Notably, it is not necessary to calculate the numbers of microspheres per unit blood--although this number may also be determined.
Later, when the animal's tissue samples are harvested, each tissue sample obviously contains but a minute fraction of the millions of injected microspheres that are now lodged within, and blocking, of a corresponding number of minute capillaries of the billions of such capillaries within the animal's entire body. The intensity of microspheres within the harvested tissue may likewise be expressed in terms of units radioactivity (i.e., radioactive intensity) per unit measure of volume or of weight. Dividing the harvested radioactive intensity by the injected intensity causes the specific radioactive strength of the microspheres to cancel out of the equation, and the volume blood flow (normally expressed in ml/min/gm) reaching the organ of interest is directly determined. Prior art dye-elution microspheres, which will be later discussed, work on the same principle.
Dye-colored microspheres are better adapted to long term quantification than are radioactive microspheres once a microsphere is (quantitatively) color-dyed as it then holds the dye, without appreciable change, during all conditions of storage and passage through the bloodstream.
2.4.3 Limitations of Conventional Radioactive Microspheres
The main (but not the only) problem with radioisotope-labeled microspheres is shelf life. In order that the decay events from the radioisotopes should be detectable during the lapse of a reasonable period of time, the radioisotopes must have short half-lives. A radioisotope lodged on a "radioactive microsphere" commonly has a radioactivity "half-life" that is as short as several days and no longer than a few weeks or months; the intensity of the radioactive emission from the radioactive microsphere decreasing by half with each passing of the "half life" period. Since radioactivity decays with time, it becomes necessary to inject larger and larger numbers of aged microspheres to permit that the microspheres should still be reliably detectable.
A supply of radioactive microspheres ages even while they are on the shelf. Radioactive microspheres thus have a time limited shelf life, which adds a cost factor to their use. The problem of decreasing radioactive intensity does not end with injection into an animal. Care must also be taken not to let too much time go by before harvesting and analyzing the tissue samples or there may be insufficient activity to determine low fluid flows due to the `noise` threshold of a typical gamma counter used for measurement of radioactivity. Constant replenishment, inventory management, and renewal of microspheres used in, principally, biological experimentation is an onerous laboratory task. If not performed diligently experimental schedules may be disrupted. Although due precautions are taken in transport and storage of radioactive microspheres, the constant flux of newly produced radioisotopes from manufacturer to laboratory, the controlled storage of radioisotopes still suitable for experimental use, and the long term of radioisotopes no longer suitable for use but still sufficiently radioactive so as to be unsuitably released into the environment, all involve a biohazard.
The present invention will be seen to avoid this problem entirely; all isotopes being distributed, stored, used, optionally stored again, and re-shipped in a non-radioactive form over an indefinitely long time period with an arbitrarily long duration in each phase. Meanwhile, the high sensitivity and specificity of radioactive labeling will be seen to be preserved.
Still other problems and disadvantages associated with radio-isotope labeled microspheres will be seen to be eliminated or substantially abated.
First, the high start-up costs of using radioactive isotopes will be seen to be avoided. These costs commonly include special government licensing, and purchase and maintenance of each of a gamma counter to measure radioactivity, shielding to protect laboratory workers from radiation exposure, and complex storage facilities. There is typically a high minimum "per order" cost of equipments from manufacturers. These high costs severely limit the use of radioisotope-labeled microspheres in blood flow measurement, generally restricting its use to large laboratories and medical centers.
Second, because of the half-lives of their contained radioisotopes, radioactively-labeled microspheres have a limited shelf life typically ranging from weeks-to several months.
Third, because of the short half-lives of many radioisotopes, radioactively-labeled microspheres are typically usable only in experiments of durations that are no more than a few weeks or months.
Fourth, commercially available automated gamma-ray counting equipment is NaI-based. Sodium-iodine (NaI) crystals provide a low-cost, sensitive gamma-ray counting system with intrinsically poor spacial resolution. As a result, researchers are limited in the number of different radioactive microspheres that can be accurately measured per sample, due to overlap between the emission energies of available radiolabels. Typically, researchers are limited to five radiolabels. Increasing the number of radiolabels measurements is done only at a significant loss in sensitivity and specificity. (The measurement of the separate radioactively-determined blood flows is performed by mathematically-based techniques. Namely, "matrix-inversion" analysis is performed to remove the known "spill-over" between the emission spectra of various emitting species. A "cross-over" matrix is mathematically solved. These techniques are similar to the spectrographic analysis of a palette of dye-colored microspheres.)
Fifth, laboratory workers using radioactively-labeled microspheres are exposed to radiation danger. The radioactively-labeled microspheres are especially dangerous if they enter into the human body by ingestion, respiration, or accidental injection. They are so small, and so numerous, so as to be incapable of removal. Accordingly, the costs, and risks, involved in minimizing radiation exposure can be substantial.
Sixth, licenses are required form various local, state, and national Governmental regulatory agencies in order to transport, possess, use, and dispose of radioactive materials, including radioactively-labeled microspheres.
Finally, and perhaps most critically, disposal of the experimental animals poses significant problems, both logistically and financially. Because the entire animal carcass remains radioactive for some time after use, and must be placed in a special low level radiation dump, to which dumps there is increasing public antipathy. The cost of disposal is becoming prohibitively high, recently ranging to as high as $750 U.S. or more per animal.
2.4.4 The Earliest Measurements of Blood Flow With Colored Microspheres
Colored microspheres are primarily relevant to the present invention for showing (i) the increased flexibility in experimental procedures that may be realized when radioisotope-labeled microspheres need not be timely extracted and measured, and (ii) the powerful ways by which, adequate time being had with no danger from radiation, the microspheres can be chemically and even mechanically tailored on a particular experimental protocol.
In 1967, polystyrene latex, radioactively-labeled microspheres (RM) were introduced for the measurement of regional perfusion. See Rudolph A M, Heymann Mass.: The circulation of the fetus in utero: Methods for studying distribution of blood flow, cardiac output and organ blood flow. Circ Res 1967; 21:163-184.
One year later, Makowski, et al., introduced a blood withdrawal technique for the quantifying of regional blood flow. See Makowski E L, Meschia G, Droegemueller W, Battaglia F C: Measurement of umbilical arterial blood flow to the sheep placenta and fetus in utero: Distribution to cotyledons and the intercotyledonary chorion. Circ Res 1968; 23:623-631.
In 1969, Domenech, et al., first validated the use of radioactive microspheres (RM) for the measurement of regional myocardial blood flow (RMBF). See Domenech R F, Hoffman J I E, Noble M I M, Saunders K B, Henson J R, Subijantos: Total and regional coronary blood flow measured by radioactive microspheres in conscious and anesthetized dogs. Circ Res 1969; 25:581-596. Thereafter, this method has become the standard technique for the measurement of RMBF in various experimental settings. However, due to the precautionary measures needed to minimize radiation exposure, use of RM is restricted to specially licensed laboratories. As mentioned above, storage of the radioactive microspheres, as well as disposal of radioactive waste, is expensive and presents a health and environmental hazard.
To avoid some of these limitations inherent to the RM method, U.S. Pat. No. 4,616,658 to Shell, et al. for NON-RADIOACTIVELY LABELED MICROSPHERES AND USE OF SAME TO MEASURE BLOOD FLOW Describes a method for measuring RMBF using non-radioactive, colored microspheres (CM). Later, Hale, et al., described a similar technique. See Hale S L, Alker K J, Kloner R A: Evaluation of non-radioactive, colored microspheres for measurement of regional myocardial blood flow in dogs. Circulation 1988; 78:428-434.
According to the techniques of Shell, et al., and of Hale, et al., microspheres may be (i) labeled with colored dyes, and (ii) subsequently visually identified and counted after recovery from digested tissue, either after separation therefrom or while still trapped in the tissue's capillaries. Shell, et al. also describe labeling microspheres by linkage to enzymes, particularly plant enzymes, and, after extraction from tissue, measuring the density of enzyme-linked spheres by a measurement of colorometric density which is indicative of enzyme activity.
In the previous techniques using non-radioactively-labeled dye-colored microspheres (CM), tissue samples that have trapped, or captured, microspheres from the circulating blood of a live animal are surgically harvested after euthanasia of the animal; and are then digested by a combination of enzymatic and chemical methods. Aliquots of the microspheres trapped within a given sample are then counted in a hemocytometer by an investigator using light microscopy, or, in the case of enzyme-linked microspheres, by measurement of colorometric density to determine enzyme activity.
There are, however, significant limitations. to these previous counting techniques. First, RMBF is extrapolated from only a small aliquot of the dye-colored microspheres (CM) actually trapped within the sample, thereby entailing a substantial statistical error in RMBF calculations. Second, the use of a maximum of only three different colors (in the same experiment) has been validated in the literature, and then in only a small number of samples, whereas it is clearly desirable to be able to make more than three measurements of RMBF in many common experimental protocols. Third, there was considerable variation in the diameter of the CM used in previous studies, as admitted by Hale et al. Fourth, the prior methods require substantial time for the tedious counting of individual dye-colored microspheres. Automation for optical counting is expensive, typically $40-50 K U.S. circa. 1993. Fifth, in preliminary experiments, the inventors of the present invention found it almost impossible to distinguish visually the nine (9) commercially available microsphere colors in the reddish background of digested myocardium.
Recently, still another alternative non-radioactive method for measuring RMBF was developed by Morita, et al. using X-ray fluorescence excitation of microspheres loaded with elements of high atomic number. See Morita Y, Payne B D, Aldea G S, McWattes C, Huseini W, Mori H, Hoffman J I E, Kaufmann L: Local blood flow measured by fluorescence excitation of non-radioactive microspheres. Am J Physiol 1990; 258:H1573-H1584. So far, only two different labels have been reported to have been validated by comparison to radioactive microspheres (RM) after intracoronary injection in two dogs. The method of Morita, et al. could be hampered by leaching of the label from the microspheres over time. Another disadvantage is the need of a sophisticated and extremely expensive equipment for X-ray excitation and fluorescence detection which is not commercially available.
The previous blood flow analysis methods employing dye-colored microspheres, including the method of Morita, et al., require that the numbers of microspheres per unit portion of a recovered tissue sample should be determined. Because the numbers of microspheres introduced within the blood [typically five to ten million (5-10.times.10.sup.6)], and captured within the capillaries of the tissue, are large in the counting techniques, the actual numbers are commonly only estimated by statistical sampling, which induces measurement error. Worse, even the determination of the numbers of dye-colored microspheres that are within minute sub-samples is tedious and expensive, involving in the methods of Shell, et al., and of Hale, et al., manual or semi automated observations through a microscope.
In order to circumvent these limitations, it would be desirable if a new method of producing and/or using microspheres, and of measuring RMBF therewith, could support both (i) easy tissue processing (i.e., digestion) and (ii) quantitative, automated, and easy counting of every microsphere within an individual sample. Such a new method would desirably be both economical and validated by a rigorous comparison to RM over a range of RMBF from 0 to 10 ml/min/gm on many hundreds, or thousands, of individual myocardial samples. If such a method were to be suitably economical, reliable, easy to use, and devoid of significant drawbacks, then it might find general use in the measurement and analysis of diverse fluid flow and fluid mixing problems other than only medical problems.
2.4.5 Blood Flow Measurement as Taught in the Certain Previous Patents of Assignee Triton Technology, Inc.
The predecessor patents listed in section 2.3 above, assigned to Triton Technology, Inc. of San Diego, Calif., teach advanced dye-colored microspheres, and the use thereof in blood flow measurement.
The methods of the related patents replace the "counting" of the numbers of non-radioactively labeled microspheres during a use of such microspheres in fluid flow analysis with, instead, a direct measurement of the amount of a colored, non-radioactive, dye that is carried by such microspheres. The dye is removed from the recovered microspheres by elution or by simply dissolving the spheres. The solvent is then analyzed for dye content by absorbance or fluorescence spectrophotometry. Measurement of the amount of dye accomplished directly by spectrographic methods is much easier and faster than counting the numbers of microspheres, and accounts for all the microspheres in the sample, and not just a small aliquot.
In particular, the (i) microspheres of the prior patents are dyed with a color for which the quantitative photometric absorption or emission (fluorescence) spectrum is uniquely identifiable, (ii) the labeled and dye-colored microspheres (CM) so created are introduced in a fluid flowing into a volume serving as a reservoir of such fluid, (iii) after the introduction of the CM the concentration of dye within the fluid (concentration being the amount of dye per unit portion of fluid) is determined, (iv) the CM are recovered from a complete sample, not a small aliquot of the sample volume into which the fluid containing the introduced CM has flowed, (v) the colored dye is eluted or dissolved from the recovered CM with a solvent, and (vi) the recovered dye is quantified by a spectrophotometric procedure. The relative amplitude of the photometric spectrum of the recovered dyes gives a quantitative indication of the concentration of the dyes within the sample. The concentration of dye that was within the original fluid is normally similarly determined, i.e. by spectroscopy. The ratio of these two concentrations indicates the flow of the fluid within which the CM were previously resident into the volume relative to the overall flow of fluid.
Fluorescent dye spectrum analysis offers some apparent advantages for CM applications, when compared to absorbance spectra analysis. Fluorescent dyes emit light isotopically and can be read off-axis (i.e., at 90.degree.) from the excitation source. Because reflection of the excitation light is minimized at high angles, this serves to minimize noise in the form of extraneous light. The complete emission spectra can be de-convolved mathematically to analyze the areas under the waveforms if the. increased sensitivity of "matrix inversion" peak analysis is required. Thus emission dyes may sometimes offer increased sensitivity over absorption dyes. However, statistical requirements for the minimum number of spheres required for a `significant` measured value in a given tissue sample (typically 400 spheres per-sample) partially offset the major advantage in the sensitivity with which fluorescent, as opposed to absorption, dyes may be detected. The primary advantage of fluorescent dyes appears to be their (i) excitation with a distinctive frequency of radiation in order to fluoresce, and (ii) their potentially sharper, or narrower `peaks`, both making it theoretically possible the use wider palettes of non-interfering colors than with purely absorption dyes.
When the (ii) introducing of the CM is into the circulating blood of a live animal, and when the (iii) determining is of the concentration of dye within the circulating blood, and when the (iv) recovering of the CM is from harvested animal tissue and blood by process of tissue and blood processing, then the (vi) measuring serves as a quantitative indication of the concentration of the dye in the harvested tissue and blood, and thus of the flow of the blood within which the CM, and the dye, were contained to the harvested tissue.
The (i) coloring is typically of each of several different types of microspheres: the microspheres of each type becoming labeled with an associated one of a plurality of different colors. The quantitative photometric spectrum of each color is both a) individually uniquely identifiable, and b) distinguishable from the photometric spectrum of all other colors. When the (ii) introducing, and the (iv) recovering, are of the several different types of microspheres--either of which steps may transpire separated in time and/or space, and may be repeated--then the (vi) measuring in a spectrophotometric procedure is of the composite quantitative photometric spectra of the several recovered dyes. Accordingly, an expanded method includes the additional step of (viii) mathematically analyzing, or de-convolving, the composite spectra to account for spectral overlap between the individual spectra of the several dyes at each of several specific wavelengths, namely the wavelengths of the individual peak absorption (or emission) of the several dyes. The mathematical analysis preferably transpires by one of several computerized mathematical processes, including matrix inversion. Each such individual spectrum is a quantitative indication of the concentration of the dye associated with each individual type of labeled CM, and a corresponding indication of the flow of that (those) fluids within which each type of CM was previously resident into the volume.
For emission spectrometry (as is taught in the predecessor patents) the (vi) measuring and (viii) analyzing steps are both easy and susceptible of automation. Despite their relative ease and simplicity, the steps are fully capable of accurately simultaneously determining the absolute, and relative, abundances of a number of different types of dye which are within a corresponding number of different types of CM. These different types of CM may be of different sizes, densities, shapes, or surface characteristics--each of which may have correspondingly different propensities to lodge within tissue or other material (such as soil) contained within the volume. The different types of CM may have been placed within several different flowing fluids that were subsequently mixed. The different types of CM may have been placed within the same stream of flowing fluid at different times. Accordingly, just one automated photometric analysis readily yields an abundance of temporal and spatial information regarding fluid(s) flow(s) and fluid mixing. Such abundant information is, in particular, eminently suitable for medical blood flow analysis including regional myocardial blood flow (RMBF) analysis, but is not so limited.
The methods of the predecessor patents were validated by its production of quantitative results that are. in close correlation to RMBF measured by 15 .mu.m diameter radioactive microspheres after intracoronary injection in 4 pigs (r=0.98), and after intra-atrial injection in 4 dogs (r=0.97). The methods of the related predecessor patents are (i) fast, (ii)-easy, (iii) susceptible of automation and (iv) cost-effective, while avoiding all-problems related to radioactivity. However, tissue digestion is still required.
When referring to microspheres, radioactive microspheres and dye-colored microspheres are called "types". Each particular radioisotope, having a particular emission energies, that serves tc radio-label microspheres of the radioactively-labeled type (i.e., RM) is spoken of as creating a "species" of that type. Similarly, each different color of dye-colored microspheres, or CM, is spoken of as being a particular "species" of CM.
The present invention will later be seen to improve upon the very nature multiple-species type of microspheres, and to contemplate automatic accurate measuring of this new type of microspheres by neutron activation analysis, and without the necessity of digesting anything, or eluting the marker label from the microspheres.
2.4.6 The Desire and Need to Conduct Many Blood Flow Tests Simultaneously and/or in Series Sequence Before Subletting a Laboratory Animal to Euthanasia and Harvesting its Tissue
When an expensive laboratory animal is subjected to blood flow analysis in order to evaluate the effects of various medical regimens, therapeutic and otherwise, there is a strong desire and need, based on efficiency of labor and reduction of cost, to accomplish as much investigation at one time as is feasible. A first injection of microspheres (of any type and species) typically later serves, when the animal's tissue is harvested, as a bench mark of normal blood circulation to the organ of interest, and serves as a baseline or reference point. After this bench mark injection the experimental protocol begins. For example, a coronary artery perfusing a portion of the heart of an animal might be partially or completely clamped, simulating a coronary event ("heart attack"). Another, second, injection of microspheres (of another type and/or species) generally serves to show, when later detected in harvested tissue, a diminished (or non-existent) blood flow, to the portion of the heart perfused by the clamped or partially clamped coronary artery. Other portions of the heart perfused by other coronary arteries will typically show no, or only slight, changes in blood flow during the same intervention. Similar injections at a number of subsequent times generally serves to define, when correlated with procedures and interventions performed on the animal and/or the administration of drugs to the animal, exactly how the animal's target organ is being perfused under several successive steps of an experimental protocol conducted over a period of time.
In order to extract from the selected harvested tissue, and from the blood samples, the record of the various blood flow circulations at the various times of the successive injections, it is necessary to separately evaluate the presence (or absence) of each different type and species of microspheres as were injected into the animal upon successive times. With radioactive microspheres, some 7-8 overlapping types of radioisotopes in common use each produce a distinct signature--permitting thereby an experimental protocol having up to 7-8 interventions and/or measurements at successive times. Of course, in accordance with the half lives of the radiolabels of the radioactive microspheres, the experimental protocol, regardless of the number of steps, should always be terminated, and the radioactivity analyzed before the shortest half-life isotope becomes too Weak to be useful.
Dye-labeled colored microspheres, or CM, are stable, and will support experimental protocols of long time duration. Presently, in the methods of the predecessor patents, typically more than five absorbance, and even more fluorescent, dye-colored microspheres may be commonly reliably separately distinguished in the presence of each other. The search goes on for palettes of even more dyes that are. individually distinguishable from one another in their spectrums of absorption or emission when a number:of such dyes are all mixed together.
In general, a desirable characteristic sought for dyes used to dye CM is a single very sharp peak absorbance or emission at a wavelength suitably displaced from all other dyes of the pallet. The spectrum of thousands of dyes have already been examined for these characteristics, and the search continues. However, with existing spectrometer sensitivities, and with analytical software programs of tractable size and execution times, which, most importantly, produce accurate quantitative results in the analysis of the individual spectral outputs of several different dyes (derived from species of CM) mixed together, it has, to date simply not been possible, to identify compatible dyes, and families of dyes, that number more than approximately one dozen. Accordingly, the number of separate steps in experimental protocols using CM is currently, circa 1993, limited to a dozen or less, and is more commonly and routinely (exotic dyes not being used), limited to about seven.
Researchers would prefer that the (i) duration of their experiments, and (ii) the number of intervention steps, in an experimental protocol should quite literally be unbounded. Although a total lack of limits may not be possible, it would be useful if some scheme could be developed to permit the individual detection in harvested tissue of large numbers (more than 15-20) of species of colored microspheres--which CM are not subject to deteriorate over time. Such detections would obviously permit that effective, extensive, multi-step experimental protocols could ensue over indefinitely long periods of time before it became necessary to perform euthanasia on the animal, and harvest its tissue, to evaluate the effect on blood flow of the step-wise procedure. Accordingly, blood flow experiments of considerable complexity, and many intervention steps, can be performed before the animal needs be subject to euthanasia, and its tissue harvested.
2.4.7 Efficiency Issues When Conducting Great Numbers of Blood Flow Measurements by Methods as are Taught in the Predecessor Patents
The flow measurement methods taught within the related patents although a step forward over prior art radioactive microspheres and measurement methods--require certain steps that previous radioactivity-measuring methods using radioactive microspheres did not require. In previous radioactive methods; the abundance of radioactive microspheres (RM) in a unit sample of the harvested tissue is directly determinable by measurement of the level of gamma radiation emitted by each species of RM present within the sample. This measurement is performed without extracting the microspheres from the harvested tissue sample nor, for the matter, without extracting the radioactive substance from the RM.
When dye-colored microspheres (CM) are used instead, as is taught within the related predecessor patents, then a necessary and ultimate step is the quantitative assessment, by emission or absorbance spectroscopy, of the amount of dye that is within each species of the collective CM that are within the harvested tissue sample. This particular measurement step may be roughly as easily performed as are radioactivity measurements, and may be performed at roughly the same, or at lessor, cost than are radioactivity measurements.
However, in order to extract the dye(s) from the collective microspheres that are within the harvested tissue sample, certain additional process steps are required before the measurement step. First, the harvested tissue qroup must be digested. Next, the dye-colored microspheres (CM) must be recovered from the digestate, normally by centrifugation and filtration. Finally, the dye(s) must be eluted or dissolved from the recovered CM.
Of these three additional steps, the second is by far the most labor intensive, and is therefore the most expensive. Both the digestion of the tissue, and the eluting of the dye from the recovered CM are chemical processes requiring only the addition of appropriate reagents. The recovery of the CM by centrifugation or filtration is, however, a time-consuming and attention-demanding process that must be carefully performed.
Accordingly, it would be useful if some improvement could be made to the multi-step method of the related patents in order to (i) simplify and/or (ii) automate the. separation of the CM form the digestate, and/or the dye form the separated CM, or else (iii) eiimina e the requirement entirely. An improvement to CM would logically permit direct measurement of the CM that are within the harvested tissue sample in steps that were either (i) reduced in number, (ii) simplified, and/or (iii) automated, or semi-automated. Otherwise, a new type of microsphere is required, and that is the subject of the present invention.